The growing epidemic of obesity, insulin resistance, and Type 2 diabetes requires new strategies for prevention and treatment. We recently discovered a structurally novel, bioactive family of lipids, branched Fatty Acid esters of Hydroxy Fatty Acids (FAHFAs), which are synthesized in vivo. A subclass of these, Palmitic Acid esters of Hydroxy Stearic Acid (PAHSAs), have anti-diabetic and anti-inflammatory effects. In insulin-resistant people, PAHSA levels in serum and adipose tissue are reduced compared to insulin-sensitive people, and levels correlate highly with insulin sensitivity. In insulin-resistant mice, PAHSA administration lowers blood glucose, stimulates GLP-1 and insulin secretion, improves glucose tolerance and reduces pro-inflammatory cytokine levels in adipose tissue. In vitro, PAHSAs augment insulin-stimulated glucose transport in adipocytes and glucose-stimulated insulin secretion from human pancreatic islets. There are 8 PAHSA isomers that differ by the position of the ester bond. PAHSA concentrations are regulated under physiologic (fasting) and pathophysiologic (high-fat diet) conditions in numerous tissues. The discovery of these novel lipids indicates the existence of unknown biochemical pathways for their synthesis and degradation. The overall goal of this proposal is to identify the enzymes that regulate the biosynthesis and degradation of PAHSAs, and to determine the relative importance of synthesis, degradation and secretion in controlling PAHSA levels in physiologic and pathophysiologic states. We have already made tremendous progress with the identification of the first PAHSA hydrolase; the development of a robust protocol that enables the biochemical purification of PAHSA biosynthetic enzymes from cells and tissues; and in vivo methods to measure PAHSA biosynthesis, degradation and secretion in awake mice. These studies will enable us to determine the relative contributions of these processes to PAHSA regulation and which mechanisms are responsible for lowering PAHSA levels in insulin-resistant states. In this application, we will integrate biochemistry, genomics, analytical chemistry and physiological experiments to identify, validate and characterize PAHSA regulatory enzymes, and to define the biochemical pathways that are responsible for controlling endogenous PAHSA levels. Because of the beneficial biologic effects of PAHSAs, these studies have the potential to reveal new targets to prevent and treat type 2 diabetes.